7. Project Summary/Abstract Inteins are phylogenetically diverse protein splicing elements that attract attention for their mechanisms of autocatalysis and application in research, biotechnology and medicine. Interest in these elements further stems from their ability to act as mobile genetic elements at the DNA level and the role they play in genome evolution. Intein research exists at the crossroads of the disparate disciplines of protein chemistry, structural biology, genetics, molecular evolution and biotechnology. The autocatalytic peptide cleavage and ligation reactions make inteins useful tools in modern chemical biology, whereas intein presence within proteins critical to cellular processes raises provocative questions about their potential function in nature. In the past funding period, we made considerable progress in functional and structural characterization of inteins, most notably that some act as regulatory sensors that respond to environmental cues. We also solved the crystal structure of an intein complexed with cisplatin, a potential novel antimicrobial agent. We propose the following three specific aims, based on discoveries made in the past funding period: In the first aim, we will characterize phage intein distribution and dissemination, and test the hypothesis of phage inteins as environmental sensors. We will also investigate the dynamics of intein mobility and pursue the observation that ATPases are intein sinks. In the second aim, we will probe the function of inteins from microbial pathogens, by investigating the Mycobacterium tuberculosis intein in SufB, an iron cluster assembly protein, as a metal ion sensor. We will also examine differential regulation of the two inteins in mycobacterial DnaB, a replicative helicase, and determine the role of the spliceosomal Prp8 intein in RNA splicing of fungal pathogens. In the third aim, we will analyze intein mechanism for applications in biotechnology, by developing a thiazoline-based intein switch for bioseparation and intracellular pH sensing. We will also characterize potent intein inhibitors that prevent the growth of M. tuberculosis, as mechanistic probes and potential antibiotics. Once again, we are taking collaborative, interdisciplinary approaches, which combine bioinformatics, biochemistry, genetics and microbiology with structural biology. In this way, we will enhance our understanding of the structure, function and evolution of inteins, as a means to exploit them as novel reagents in biotechnology and as potential antimicrobial drug targets.